1. Field of the Invention
The present invention relates to a technique for fluorescence analysis, and more particularly to a technique for improving a signal-to-noise ratio in detection of faint fluorescence emitted by a single molecule or of faint fluorescence emitted by a small number of molecules.
2. Description of the Related Art
In order to detect a target substance such as a deoxyribonucleic acid (DNA) and a protein, the following method has been widely used. That is, a target substance is fluorescently labeled and irradiated with predetermined excitation light such as a laser beam, and fluorescence generated by the irradiation is detected. As a nucleic acid analysis device, a new technique for determining a base sequence of a DNA and a base sequence of a ribonucleic acid (RNA) has been developed.
In a method using electrophoresis that is typically used, a reverse transcription reaction is performed on a DNA fragment or an RNA sample, which is used to determine a sequence, to prepare a synthesized complementary DNA (cDNA) fragment sample; a dideoxy reaction is performed through a known Sanger method; the electrophoresis is performed; and a molecular weight separation and a molecular weight distribution are measured and analyzed.
In recent years, as disclosed in “P.N.A.S. 2003, Vol. 100, pp. 3960-3964” (Non-Patent Document 1), a method for fixing a DNA or the like to a substrate and determining a base sequence of the DNA or the like has been proposed. This method is generally called “sequencing by synthesis”. In this method, sample DNA pieces to be analyzed are randomly captured by the surface of the substrate on a molecule basis; bases are elongated on a single base basis or on a several-base basis; and the results of the elongations are detected through a fluorescence measurement to determine a base sequence. In this method, there is a possibility that the base sequence can be determined for each DNA molecule. Therefore, it may be unnecessary that a sample DNA be purified and amplified by cloning, a polymerase chain reaction (PCR) or the like. Therefore, it can be expected that a genomic analysis and a genetic diagnosis are accelerated. In Non-Patent Document 1, a solution is exchanged for cleaning in order to reduce background noise and improve a signal-to-noise ratio.
In “PNAS 2005, Vol. 102, pp. 5932-5937” (Non-Patent Document 2), a method for determining a DNA sequence using a stepwise elongation reaction is disclosed.
In “Optical Application Technology, Material Encyclopedia, Chapter 2, Section 1, Page 8, “Optical cleaning”, Sangyo Gijutsu Service Center” (Non-Patent Document 3), photo-cleaning by ultraviolet excimer lamps is disclosed as a cleaning method used in another technical field. The photo-cleaning by ultraviolet excimer lamps is performed for an organic substance attached to the surface of a substrate in a process of manufacturing a liquid crystal panel. Light, which is emitted by an excimer lamp and has a wavelength of approximately 172 nm, has photon energy larger than bonding energy of a large number of covalent bonds present in the organic substance. The organic substance is therefore decomposed and vaporized, and the surface of the substance can be cleaned.
In a method disclosed in JP-A-2006-153639 (Patent Document 1), a region present on a substrate, in which a probe is not present, is irradiated with light to eliminate fluorescence emitted by a fluorescent dye that is not captured by a probe.
In U.S. Pat. No. 7,329,492 (Patent Document 2), a method for determining a DNA sequence is disclosed. In the method, an enzyme is fixed to a substrate, and the DNA sequence is determined using fluorescence resonance energy transfer (FRET).
Non-Patent Document 4 “Daniel Axelrod et al. ‘Total Internal Reflection Fluorescence Microscopy Interactive Java Tutorials Evanescent Field Penetration Depth’. [online]. Olympus America Inc. [retrieved on 2009-02-24]. Retrieved from the Internet: <URL: http://www.olympusmicro.com/primer/java/tirf/penetration/index.html>.” describes about evanescent light. An electromagnetic wave (light) having a wavelength of λ is incident on an interface between an incident-side medium having a refractive index of n1 and an outgoing-side medium having a refractive index n2 at an incident angle of θ. In this case, the following expression is established: θc=sin−1 (n2/n1). The symbol θc is called a critical angle (sin−1 is an inverse function of sine). When the incident angle θ is equal to or larger than the critical angle θc, the electromagnetic wave is totally reflected by the interface between the incident-side medium and the outgoing-side medium. In this case, an electromagnetic field is generated on the side of the outgoing-side medium with respect to the interface. The intensity of the electromagnetic field is reduced exponentially with distance from the interface. The electromagnetic field is called an evanescent field (light). A relative intensity (E) of the evanescent field is a function of the distance (z) from the interface. When the evanescent field is present on the interface (z=0), the relative intensity (E) of the evanescent field is 1, which is the maximum value. The relative intensity of the evanescent field is represented by the following formula: E=exp(−z/d), where d is determined based on the wavelength, the incident angle and the refractive indexes. The value d is called a penetration depth. It is known that the penetration depth d is represented by the following formula: d=λ/(4π(n12× sin2θ−n22)1/2), where n1 is the refractive index of the incident-side medium; n2 is the refractive index of the outgoing-side medium; θ is the incident angle; and λ is the wavelength.